Pi-interaction tuning of the active site properties of metalloproteins.
The influence of pi-interactions with a His ligand have been investigated in a family of copper-containing redox metalloproteins. The Met16Phe and Met16Trp pseudoazurin, and Leu12Phe spinach and Leu14Phe Phormidium laminosum plastocyanin variants possess active-site pi-contacts between the introduced residue and His81 and His87/92 respectively. The striking overlap of the side chain of Phe16 in the Met16Phe variant and that of Met16 in wild type pseudoazurin identifies that this position provides an important second coordination sphere interaction in both cases. His-ligand protonation and dissociation from Cu(I) occurs in the wild type proteins resulting in diminished redox activity, providing a [H(+)]-driven switch for regulating electron transfer. The introduced pi-interaction has opposing effects on the pKa for the His ligand in pseudoazurin and plastocyanin due to subtle differences in the pi-contact, stabilizing the coordinated form of pseudoazurin whereas in plastocyanin protonation and dissociation is favored. Replacement of Pro36, a residue that has been suggested to facilitate structural changes upon His ligand protonation, with a Gly, has little effect on the pKa of His87 in spinach plastocyanin. The mutations at Met16 have a significant influence on the reduction potential of pseudoazurin. Electron self-exchange is enhanced, whereas association with the physiological partner, nitrite reductase, is only affected by the Met16Phe mutation, but kcat is halved in both the Met16Phe and Met16Trp variants. Protonation of the His ligand is the feature most affected by the introduction of a pi-interaction.
Yanagisawa
Sachiko
S
Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
Crowley
Peter B
PB
Firbank
Susan J
SJ
Lawler
Anne T
AT
Hunter
David M
DM
McFarlane
William
W
Li
Chan
C
Kohzuma
Takamitsu
T
Banfield
Mark J
MJ
Dennison
Christopher
C
D042422:Achromobacter cycloclastes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D032492:Dryopteris / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004583:Electrons; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D008024:Ligands; D008667:Metalloproteins; D003300:Copper